Question

Please write an essay for the following information for the Freeze fracture technique (may use online sources):

Purpose - Brief description (one to two sentences) of the purpose of this method (ie. what is the general purpose for this technique? When would it be used?)

Background – This section will include a description of the concepts and theories that are needed to understand this technique.

Method description – this section will include an overview of the steps involved in carrying out the technique. This does not need to be as detailed as you would expect to find in a publication, but rather should include more of an outline of the experimental flow.

Advantages/limitations of the technique – This section should include at least 1 advantage and 1 limitation of the technique compared to other techniques.

Answer 1

The freeze-fracture technique consists of physically breaking apart (fracturing) a frozen biological sample; structural detail exposed by the fracture plane is then visualized by vacuum-deposition of platinum–carbon to make a replica for examination in the transmission electron microscope. The four key steps in making a freeze-fracture replica are

(i) rapid freezing,

(ii) fracturing,

(iii) replication and

(iv) replica cleaning.

In routine protocols, a pretreatment step is carried out before freezing, typically comprising fixation in glutaraldehyde followed by cryoprotection with glycerol. An optional etching step, involving vacuum sublimation of ice, may be carried out after fracturing. Freeze fracture is unique among electron microscopic techniques in providing planar views of the internal organization of membranes. Deep etching of ultrarapidly frozen samples permits visualization of the surface structure of cells and their components. Images provided by freeze fracture and related techniques have profoundly shaped our understanding of the functional morphology of the cell.

Use of freeze fracturing in biology-

Cell membranes consist of phospholipids and attached or embedded proteins. Membrane proteins play vital roles in the metabolism and life of the cell. You cannot use ordinary microscopy to visualize or characterize adhesion proteins, transport proteins and protein channels in the cell membrane. Using electron microscopy and a technique called "freeze fracture," which splits frozen cell membranes apart, allows visualization of the membrane structure and the organization of proteins within the sea of phospholipids. Combining other methods with freeze fracturing not only helps us to understand the structure of different cell membranes and membrane proteins, but allows for the visualization and detailed analysis of the function of specific proteins, bacteria and viruses.

How this technique is used?

Using liquid nitrogen, biological tissue samples or cells are rapidly frozen to immobilize cell constituents. Cell membranes are composed of two layers of phospholipids, called a bilayer, where the hydrophobic, or water-hating, lipid tails point to the inside of the membrane and the hydrophilic, or water-loving, ends of the lipid molecule point outward and toward the inside of the cell. The frozen sample is cracked or fractured with a microtome, which is a knife-like instrument for cutting thin tissue slices. This causes the cell membrane to split apart precisely between the two layers because the attraction between the hydrophobic lipid tails represents the weakest point. Following fracturing, the sample undergoes a vacuum procedure, called "freeze etching." The surface of the fractured sample is shadowed with carbon and platinum vapor to make a stable replica, which follows the contours of the fracture plane. Acid is used to digest organic material adhering to the replica, leaving a thin platinum shell of the fractured membrane surface. This shell is then analyzed by electron microscopy.

Freeze fracture describes the technique of breaking a frozen specimen to reveal internal structures. Freeze etching is the sublimation of surface ice under vacuum to reveal details of the fractured face that were originally hidden. A metal/carbon mix enables the sample to be imaged in a SEM (block-face) or TEM (replica). It is used to investigate for instance cell organelles, membranes, layers and emulsions. The technique is traditionally used for biological applications but started to develop significance in physics and material science. Recently, freeze fracture electron microscopy, particularly freeze replica immunolabelling (FRIL), has provided new insights into the roles of membrane proteins in dynamic cellular processes.

Applications: To determine the structure of cells and their surface at 2 nm resolution. This technique is especially suited to the study of membrane structure and membrane proteins. Precious information about the surface of cells can be obtained.

Method: The sample (tissue, cells, liposomes ...) is cryo-protected and vitrified at high pressure or in liquid propane, inserted into the vacuum chamber of the instrument and broken (freeze-fracture) with a knife at -125°C. The partial sublimation of ice at -105°C (freeze-etching) reveals the surface of the sample. The fractured and etched surfaces are replicated with 1 nm platinum/carbon. The replica is transferred to am EM grid for TEM examination.

Sample requirements:10 to 50 μl of a highly concentrated sample suspension.

The concept and practical application of freeze-fracture processing of biological specimens was introduced by Steere1 over half a century ago. The early apparatus appropriated disparate components into a working self-contained unit1. The original apparatus was modified and refined into commercially available instruments in order to accommodate the critical need for remote manipulation, maintenance of high vacuum, and the evaporation of carbon and metals to produce a replica suitable for examination by transmission electron microscopy.

The major goal and rationale for the development of the freeze-fracture technique was to limit artifacts observable at electron microscopic resolution deriving from chemical fixation and processing used in conventional biological electron microscopy. Here the goal is to limit chemical fixation and to freeze the specimen with sufficient speed and frequently in the presence of a cryoprotectant in order to limit ice crystal formation and other freezing artifacts. More recently, this technique has found a resurgence of interest from molecular biologists and materials science investigators for examination of nanoparticles and nanomaterials.

Freeze-fracture and freeze-etch images exhibit a three-dimensional character and sometimes are mistaken for scanning electron micrographs. However, freeze-fracture preparations are examined by transmission electron microscopy and their major contribution to high resolution morphologic studies is their unique representation of structure/function elements of cell membranes. Freeze-fracture processing is initiated by freezing cells and tissues with sufficient speed to limit ice crystallization and/or with the use of cryoprotectant agents such as glycerol. The specimens are then fractured under vacuum and a replica is generated by evaporation of carbon and platinum over the fractured surface. The original specimen is digested from the replica which is retrieved onto a standard EM specimen grid. Another common misinterpretation of freeze-fracture images is that they depict cell surfaces. However the basic premise of freeze-fracture is that biological membranes are split through the lipid bilayer by the fracture process.

RESULTS-

The key premise of freeze-fracture image interpretation is that fracture planes pass through the lipid bilayer of membranes conferring two fracture faces, called by convention the PF-face (plasma fracture-face) and EF-face (extracellular fracture-face)

The PF-face is the half of the membrane lipid bilayer adjacent to the cytoplasm of the cell and the EF-face is the half of the membrane lipid bilayer adjacent to the extracellular milieu. The freeze-fracture technique is particularly useful for the investigation of membrane structure and the two faces are typically distinctively different with PF-faces being populated with many membrane associated particles in contrast to EF-faces which contain fewer. The cytoplasmic contents of freeze-fractured cells are typically coarse in appearance and are not particularly revealing although membrane bound organelles such as nuclei and Golgi can be readily identified. Of particular interest to investigators using this technique are specializations of membrane structure that can be interpreted for their function. These include specific distributions of membrane associated particles, ciliary membrane specializations, and intercellular junctions.

Advantages: Sample preservation, high contrast. Small membrane proteins can be visualized.

Disadvantages: Only the external surfaces and the regions where the fracture occur are visualized.

Limitations-

While freeze-fracture does provide information on the spatial organization of membranes, several limitations are associated with the technique. First, the fracture plane initiated in the sample cannot be controlled (i.e., follows a random pathway).